Identification of genes differentially expressed during water-deficit stress is an important step for developing a strategy to improve drought tolerance of cultivated, upland cotton, Gossypium hirsutum L. The objective of this study was to develop a model of the responses that lead to drought tolerance in the roots of the drought tolerant cultivar, Siokra L23. A polyethylene glycol (PEG)-induced phenotype derived from time-course measurements of photosynthesis and leaf temperature revealed a decline in the photosynthetic rate and an increase in leaf temperature between 0 and 48 h, followed by a return to initial values from 48 to 96 h. Due to the biphasic nature, these two phases were designated as response and recovery, respectively. Time-course root sugar content assessment revealed increased sucrose levels at 24 h, indicating osmotic adjustment was a component of the adaptive response. Root gene expression profiles were developed at 0, 1, 4, 24, and 96 h after stress initiation utilizing cotton-fiber-based oligonucleotide microarrays to gain insight on the cellular processes that lead to recovery. A total of 422 PEG-responsive genes were identified. Genes involved in K+, sucrose, and organic acid utilization, including two novel anaplerotic reactions feeding substrate into the TCA cycle, are proposed to be involved in root-cell osmotic adjustment. Solute accumulation was coupled to coordinate reductions in aquaporin gene expression. These observations suggested that regulation of membrane permeability to water along with cellular solute potential are primary root-localized mechanisms by which Siokra L23 adapts to PEG-induced osmotic shock.